This application is claiming a priority based on a Japanese Patent Application No. 61727/97 filed on Mar. 3, 1997. The contents of the patent application are incorporated herein by reference.
This invention relates to a modified pyrrolo-quinoline quinone (PQQ) glucose dehydrogenase (GDH). The enzyme of the invention exhibits an improved substrate specificity, thus is useful for measurement of glucose in clinical assays and food analyses.
Glucose is present in the blood and has been utilized as an important marker of diabetes. Also, in fermentation with the use of microorganisms, the measurement of glucose as growth media for microorganisms is an important parameter in process monitoring. Glucose has been assayed by enzymatic methods using glucose oxidase (hereinafter referred to as GOD) or glucose 6-phosphate dehydrogenase (hereinafter ref erred to as G6PDH). In the method with the use of GOD, however, it is necessary to add catalase or peroxidase to the analysis system, which catalyzes hydrogen peroxide formed through the glucose oxidation reaction, in order to induce the color development reaction. Further, attempts have been made to develop biosensors with the use of GOD. In these biosensors, however, the enzyme reaction depends on the concentration of oxygen dissolved in an aqueous solution. Thus they are unsuitable for samples with high glucose concentration or could provide data including errors depending on the dissolved oxygen concentration. On the other hand, G6PDH has been employed in the assay of glucose by a spectrochemical method. However, this method involves a troublesome procedure of adding a coenzyme NAD(P) to the reaction system.
Recently, the use of a novel enzyme PQQGDH has been noted as an alternative to the enzymes employed in the conventional methods for enzymatic assay of glucose. Since PQQGDH is an enzyme bearing a coenzyme bound thereto and does not require oxygen as an electron acceptor, this enzyme is expected to be applicable in the field of analysis, for example, as a recognition element of a glucose sensor. In particular, the structural gene of glucose dehydrogenase PQQGDH derived from E. coli and bearing pyrrolo-quinoline quinone as a coenzyme was known [AM. Cleton-Jansen et al., J. Bacteriol. (1990) 172, 6308-6315], and its heat stability could be improved [K. Sode et al., FEBS Lett. (1995) 364, 325-327], thus the PQQGDH of E. coli is expected to be suitable for various applications. However, the PQQGDH derived from E. coli has a problem in its substrate specificity. Namely, it reacts not only with glucose but also other saccharides [Ameyama et al., Agric. Biol. Chem. (1986) 50, 49-57].
It was previously reported that a mutated enzyme, wherein the histidine 787 residue had been spontaneously mutated into an asparagine residue in the structural gene of the PQQGDH of Gluconobacer oxydans (hereinafter referred to as G. oxydans), had a lower substrate specificity and higher reactivities with saccharides other than glucose, compared with the wild type PQQGDH of G. oxydans [AM. Cleton-Jansen et al., Mol. Gen Genet. (1991) 229, 206-212]. It is therefore believed that this site would play an important role in the substrate recognition by the PQQGDH of G. oxydans. On the other hand, the histidine 787 residue in the PQQGDH of G. oxydans corresponds to the 775 amino acid residue in the PQQGDH of E. coli, which is also a histidine residue [G. E. Cozier and C. Anthony, Biochem. J. (1995) 312, 679-685]. The PQQGDH of G. oxydans scarcely reacts with saccharides other than glucose [Ameyama et al., Agric. Biol. Chem. (1981) 45, 851-861], while the PQQGDH of E. coli can react with other saccharides. Based on these observations, the histidine 775 residue in the PQQGDH of E. coli is considered to be different in its role in determining the substrate specificity from the histidine 787 residue in the PQQGDH of G. oxydans. Accordingly, it was not possible in any way to estimate changes in the properties of the enzyme caused by substituting the histidine 775 residue in the PQQGDH of E. coli with other amino acids.